DNA damage, p14ARF, Nucleophosmin (NPM/B23), and cancer

The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF’s nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.     

Tumor stroma fosters neovascularization by recruitment of progenitor cells into the tumor bed

The tumor stroma is an active player during carcinogenesis and contains a variety of cell types such as vascular cells, fibroblasts and inflammatory cells which directly or indirectly foster neovascularization. During tumor progression stromal cells, in particular the neovasculature, acquire new characteristics distinct from their normal counterparts and display a high degree of plasticity to meet the tumor's demands. The local environment may, to some extent, shape pre-existing, tumor-resident stromal cells. However, there is accumulating evidence that new endothelial and other stromal cells are actively recruited into tumors, and that this recruitment is essential for a unique and tumor-specific proangiogenic environment.     

Design and characterization of stimuli-responsive FLAG-tag analogues and the illumination-induced modulation of their interaction with antibody 4E11

An azobenzene group containing beta-amino acid N-Fmoc-4-aminomethyl phenylazobenzoic acid was synthesized and with the exception of the C-terminal amino acid residue was substituted by solid-phase peptide synthesis into all positions of the FLAG sequence (DYKDDDDK), an octapeptide capable of specific interaction with the monoclonal antibody 4E11. The trans state of the beta-amino acid was thermodynamically more stable than the cis state. However, the molecule could be switched into the cis conformation by illumination at 340 nm. Peptides containing the artificial amino acid also became photoresponsive. In the absence of light, the spontaneous back-isomerization into the trans conformation of the photoresponsive was extremely slow (>8 h no significant increase in trans content). When illuminated with visible light (440 nm), the back-isomerization from the cis to the trans state was accelerated and occurred with a half-life of approximately 10 min. The cis form of the photopeptides was more hydrophilic than the trans form, as evidenced by differences in the retention time of the two isomeric forms in reversed-phase chromatography. Photopeptides that contained the intact sequences responsible for binding of the FLAG tag to the antibody, namely, the DYK motive at the N-terminus, showed binding to the antibody in both a dot blot immunoassay and in Biacore binding studies, albeit with lower affinity than the unmodified FLAG sequence. Peptides with a substitution in positions 4-6 showed differences in binding strength between the trans and the cis form in the Biacore studies, no such difference could be observed for the peptide with a substitution in position 7.     

The evolution and maintenance of virulence in Staphylococcus aureus: a role for host-to-host transmission?

Despite progress in our understanding of infectious disease biology and prevention, the conditions that select for the establishment and maintenance of microbial virulence remain enigmatic. To address this aspect of pathogen biology, we focus on two members of the Staphylococcus genus - Staphylococcus aureus and Staphylococcus epidermidis - and consider why S. aureus has evolved to become more virulent than S. epidermidis. Several hypotheses to explain this phenomenon are discussed and a mathematical model is used to argue that a complex transmission pathway is the key factor in explaining the evolution and maintenance of virulence in S. aureus. In the case of S. epidermidis, where skin contact affords easier transmission between hosts, high levels of virulence do not offer an advantage to this pathogen.     

Virtual fragment screening for novel inhibitors of 6-phosphogluconate dehydrogenase

The enzyme 6-phosphogluconate dehydrogenase is a potential drug target for the parasitic protozoan Trypanosoma brucei, the causative organism of human African trypanosomiasis. This enzyme has a polar active site to accommodate the phosphate, hydroxyl and carboxylate groups of the substrate, 6-phosphogluconate. A virtual fragment screen was undertaken of the enzyme to discover starting points for the development of inhibitors which are likely to have appropriate physicochemical properties for an orally bioavailable compound. A virtual screening library was developed, consisting of compounds with functional groups that could mimic the phosphate group of the substrate, but which have a higher pK_a. Following docking, hits were clustered and appropriate compounds purchased and assayed against the enzyme. Three fragments were identified that had IC₅₀ values in the low micromolar range and good ligand efficiencies. Based on these initial hits, analogues were procured and further active compounds were identified. Some of the fragments identified represent potential starting points for a medicinal chemistry programme to develop potent drug-like inhibitors of the enzyme.     

Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker

Trafficking of the pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels to the cell surface represents an important regulatory step in controlling BK channel function. Here, we identify multiple trafficking signals within the intracellular RCK1-RCK2 linker of the cytosolic C terminus of the channel that are required for efficient cell surface expression of the channel. In particular, an acidic cluster-like motif was essential for channel exit from the endoplasmic reticulum and subsequent cell surface expression. This motif could be transplanted onto a heterologous nonchannel protein to enhance cell surface expression by accelerating endoplasmic reticulum export. Importantly, we identified a human alternatively spliced BK channel variant, hSloΔ_579–664, in which these trafficking signals are excluded because of in-frame exon skipping. The hSloΔ_579–664 variant is expressed in multiple human tissues and cannot form functional channels at the cell surface even though it retains the putative RCK domains and downstream trafficking signals. Functionally, the hSloΔ_579–664 variant acts as a dominant negative subunit to suppress cell surface expression of BK channels. Thus alternative splicing of the intracellular RCK1-RCK2 linker plays a critical role in determining cell surface expression of BK channels by controlling the inclusion/exclusion of multiple trafficking motifs.     

HER2/ErbB2-induced Breast Cancer Cell Migration and Invasion Require p120 Catenin Activation of Rac1 and Cdc42

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.